Collagen has been used as a hemostyptic agent since the late sixties. Collagen is the most frequent structural protein in all mammalians. The monomeric protein of approximately 300 kDa (tropocollagen) is covalently crosslinked at specific sites. The mature protein is therefore insoluble and forms characteristic fibrils with high tensile strength. Numerous sub-classes of collagen have been described, the most common of which is collagen type I, the main collagen type in skin, tendons, bones and cornea. Collagen is a fibrous protein consisting substantially of a triple helix with a length of approximately 290 nm. Five of these triple helices (tropocollagen molecules) are staggered to form a microfibril with a diameter of approximately 3.6 nm. These microfibrils have polar and non-polar segments that are readily accessible for specific inter- and intrafibrillar interactions. Microfibrils are packed into a tetragonal lattice to form subfibrils with a diameter of about 30 nm. These subfibrils are then assembled into the collagen fibril, the basic unit of connective tissue, which has a diameter of several hundred nm and is therefore visible in a light microscope as a thin line, see reference 1. Collagen gel and collagen sponge, as produced during the manufacturing process, comprises these fibrils as the smallest units, as proved by microscopy.
Collagen may be used as a material for sealing wounds, possibly with a coating comprising a fibrin glue. Fibrin glues, i.e. the combination of fibrinogen, thrombin and aprotinin, have successfully been used therapeutically for many years for gluing tissues and nerves and for sealing surfaces when there is minor bleeding. One drawback of the fibrin glues has been that in case of major bleeding the glue is usually washed away before sufficient polymerization of fibrin has occurred. To overcome this problem surgeons have manually applied liquid fibrin glues to absorbable carriers such as collagen fleece.
Despite the impressive success of these combined applications this method has not been applied on a broad scale, due to some disadvantages. The preparation is relatively cumbersome, the method requires experience and skilled personnel, and the preparation is not readily available in cases of emergency, the time for preparation being in the range of 10 to 15 min. These factors stimulated the development of an improved product resulting in the development of a fixed combination of a collagen carrier covered with a coating of solid fibrinogen, solid thrombin and solid aprotinin as disclosed in EP 0 059 265. The product disclosed in EP 0 059 265, which has been marketed under the trademark TACHOCOMB®, is a hemostatic collagen sponge that can be applied directly to the wound. When the coating comes into contact with aqueous fluids like blood, other body fluids or saline, the components dissolve and fibrin is formed. The product is applied to the wound with a slight pressure and collagen is tightly bound (glued) to the injured surface. Hemostasis is achieved and the wound is sealed.
Beside some blood coagulation stimulating activity, the function of collagen in the hemostatic collagen sponge that TACHOCOMB® is mainly that of a carrier which adsorbs and confers mechanical stability to the coagulation preparation with which it is coated. Other advantages of collagen, in particular in the form of a sponge, are its biodegradability, its relatively high tensile strength, even in the wet state, its high resistance against the penetration of liquids and air, and its high flexibility in the wet state.
The present invention is primarily concerned with the production of a collagen sponge which may be used as a carrier for fibrinogen, thrombin and/or aprotinin, e.g., as in TACHOCOMB®. The collagen sponge may also be used directly, i.e. without a coating, as a bandage on topical injuries, for support of hemostasis, such as for prevention of rebleeding, for weak, diffuse bleeding from parenchymatic organs, for application on burns, skin grafts, decubitus or skin defects, or as a bandage on topical injuries.
In the prior art, a number of methods for preparing a collagen carrier have been suggested. WO 86/05811 discloses a weighted microsponge for immobilizing bioactive materials in motive bioreactor systems, the microsponge comprising a highly cross-linked collagen matrix. The highly cross-linked collagen matrix is prepared by milling a source of Type I, II or III collagen to yield fibers having a diameter on the order of 1 to 50 μm and a length no greater than 200 μm. The milled collagen is formed into a soluble collagen dissolved in a solvent, or an insoluble collagen dispersed in a solvent by admixture with a solvent, such as acetic acid, lactic acid, proprionic acid or butyric acid. In the case of a collagen dispersion, the mixing is accomplished with a high level of agitation using a blender, so as to produce microfibers of the collagen. Next, a weighting additive is blended with the collagen-liquid mixture and the composite mixture is formed into small droplets and solidified by freezing. A number of techniques for producing small particles are disclosed. The frozen composite is vacuum freeze-dried, the combination of freezing and drying being referred to as lyophilization. The freeze-dried collagen matrix composite is treated so as to cross-link the collagen. The collagen can be cross-linked using either chemical cross-linking agents, by severe dehydration at an elevated temperature or by a combination. The collagen matrix aimed at being resistant to collagenase and other enzymatic degradation thereby making these materials particularly suitable for culturing organisms. After washing the cross-linked collagen matrix, the microsponges may be sterilized and aseptically packaged. In the weighted microsponge, the collagen matrix has an open to the surface pore structure with an average pore size in the range of from about 1 to about 150 μm, the pores of the matrix occupying from about 70 to about 98% by volume of the microsponge. The microsponge further has an average particle size of from about 100 to about 1000 μm and a specific gravity of above about 1.05. The weighting material may be metal or alloys from metal, metal oxides and ceramics.
U.S. Pat. No. 5,660,857 discloses a process for preparing a composite comprising an insoluble protein matrix and an oleaginous material, which is useful as a material for surgical dressings and biomedical implants, and as a cosmetic material for application to the skin. The process of U.S. Pat. No. 5,660,857 comprises the steps of mixing a protein, the oleaginous material and water to form an emulsion of the oleaginous material in an aqueous dispersion of the protein, and subsequently drying or freeze-drying the emulsion to form a film or a sponge. The insoluble fibrous protein is predominantly comprised of insoluble collagen, which may suitably be obtained from bovine skin. In one embodiment, the collagen may be swollen in lactic acid prior to use.
WO 99/13902 discloses a method for producing a meningeal tissue growth matrix comprising the step of preparing physiologically compatible collagen which is substantially free of active viruses and prions. The collagen is formed into a film, a sponge, a non-woven collagen or a felt. The collagen is obtained by a process comprising cleaning skin, tendons, ligaments or bone of fat. The material is then subjected to an enzyme treatment, whereby the collagen material is swelled. The collagen material is then further swollen with an acid solution. The collagen mixture is then homogenized. The product obtained may be a matrix provided in the form of a collagen sponge, a non-woven matrix, felt or film, or a composite of two or more of the foregoing forms. A collagen sponge can be provided by adaptation of the methods for forming collagen sponges disclosed in U.S. Pat. No. 5,019,087. The sponge can be prepared by lyophilization of a collagen dispersion prepared according to WO 99/13902. The sponge density achieved is said to be about 0.1 mg/cm3 to about 120 mg/cm3. According to the disclosure of WO 99/13902, the pore size ranges from about 10 μm to about 500 μm. Laminate type of collagen sponge and collagen film are mentioned.
U.S. Pat. No. 5,618,551 relates to a non-crosslinked and potentially crosslinkable pepsintreated collagen or gelatin powder modified by oxidative cleavage in an aqueous solution, which is soluble at an acid pH and stable on storage at a temperature of below 0° C. for at least one month. The patent further relates to a process of preparing the powder, comprising preparing an acidic solution of pepsin-treated collagen, subjecting the acidic aqueous solution at room temperature to controlled oxidation, precipitating the oxidized and noncrosslinked pepsintreated collagen at an acid pH, and isolating, concentrating and dehydrating the noncrosslinked pepsintreated collaged so as to obtain it in the form of a reactive acidic powder, and freezing and storing the obtained reactive acidic powder at a temperature of below 0° C.
GB 1 292 326 discloses a method and apparatus for the preparation of collagen dispersions with a view to their applications, wherein a suspension of collagen fibers is prepared and subsequently introduced into a treatment chamber with stirring means. A sub-atmospheric pressure exists in the treatment chamber, in which the suspension is transformed into a dispersion by stirring and controlled acidification by means of a mineral or organic acid. According to the disclosure of GB 1 292 326, the preparation of spongy collagenic articles can be effected from dispersion or gels of collagen. In this context the documents refers to lyophilization and to dispersion or gels very rich in air bubbles. GB 1 292 326 further mentions a problem of controlling the introduction or the elimination of air bubbles in a satisfactory manner. The documents discloses, in two examples, a collagenic dispersion free of air bubbles with a collagen content of 2.5%, and an aerated dispersion of collagen with a collagen concentration of 2.5%, respectively.
Chemical Abstracts, Columbus Ohio, US, Vol. 98 13 Jun. 1983 No. 24 mentions a collagen obtained from animal tissues such as skin or tendon bone which has been submitted to acid treatment. The collagen is reaggregated by dialysis, during which process a net of highly birefringent crystal fibers is formed. The collagen can be shaped into 0.5 mm–2 cm sheets, or be mixed with air to form sponges, or be dispersed as a cream.